Bdellovibrio bacteriovorus uses chimeric fibre proteins to recognize and invade a broad range of bacterial hosts

Predatory bacteria, like the model endoperiplasmic bacterium Bdellovibrio bacteriovorus, show several adaptations relevant to their requirements for locating, entering and killing other bacteria. The mechanisms underlying prey recognition and handling remain obscure. Here we use complementary genetic, microscopic and structural methods to address this deficit. During invasion, the B. bacteriovorus protein CpoB concentrates into a vesicular compartment that is deposited into the prey periplasm. Proteomic and structural analyses of vesicle contents reveal several fibre-like proteins, which we name the mosaic adhesive trimer (MAT) superfamily, and show localization on the predator surface before prey encounter. These dynamic proteins indicate a variety of binding capabilities, and we confirm that one MAT member shows specificity for surface glycans from a particular prey. Our study shows that the B. bacteriovorus MAT protein repertoire enables a broad means for the recognition and handling of diverse prey epitopes encountered during bacterial predation and invasion.

experimental approaches involving differential centrifugation but yielding similar vesicle enrichments as in Supplementary Figure 8.

Enrichment 1-Percoll gradient
Two 1 litre predatory cultures of B. bacteriovorus CpoBBd0635-mCherry were grown for 24 hours at 29˚C with 200 rpm shaking (1 L Ca/HEPES buffer + 60 ml E. coli S17-1 grown in YT broth for 24 h, at 37˚C with shaking at 200 rpm + 50 ml of predatory B. bacteriovorus HD100 CpoB-mCherry culture in Ca/HEPES).The majority of cells were pelleted by centrifugation at 5,000 x g for 30 minutes.The remaining cells, cell debris and vesicles were then pelleted at 15,000 x g for 30 min.This pellet was resuspended in Percoll separation solution (24.7 ml Percoll + 5 ml 3 M NaCl + 20.3 ml H2O) and centrifuged at 17,000 x g for 30 min to separate the vesicles and prey debris from the remaining cells.A band of debris formed near the top of the Percoll gradient, and this was resuspended in 20 ml Percoll separation solution and the gradient spun again.This process was repeated one more time, and then the purified band was mixed 1:4 with H2O and spun at 5,000 x g to remove the Percoll.The resulting pellet was resuspended in 100 µl TE.

Enrichment 2; preps 2 and 3-Filtration and ultracentrifugation
One litre of a predatory culture of B. bacteriovorus CpoB-mCherry was grown for 24 hours at 29˚C with 200 rpm shaking (1 L Ca/HEPES buffer + 60 ml E. coli S17-1 or P. putida grown in YT broth for 24 h, at 37˚C with shaking at 200 rpm + 50 ml of predatory B. bacteriovorus HD100 CpoB-mCherry culture in Ca/HEPES).The majority of "contaminating" whole cells were pelleted by centrifugation at 5,000 x g for 30 minutes.The remaining cells were removed by filtering through 0.2 µm filters (Sartorius; one filter per 50 ml supernatant).Eight samples of 20 ml of this filtrate were centrifuged at 160,000 x g for 30 minutes.The supernatant was removed, and a further 20 ml filtrate was added to each tube, repeating twice further (a total of 80 ml filtrate was therefore pelleted into each of 8 tubes).All 8 pellets were pooled and resuspended in a final volume of 100 µl TE for E. coli or 400 µl TE for P. putida.

Proteomic analysis by LC/MS/MS
Proteomic analyses of the vesicle samples prepared above were carried out in the Chemistry Research Laboratory Department in the Mass Spectrometry Facility, working on a subcontract from the Oxford University Advanced Proteomics Unit Oxford UK.50 µl samples of the final vesicle samples prepared in the enrichment processes above were digested by trypsin and lysC according to the Filter-Aided Sample Preparation (FASP) method (Wisniewski et al., 2009), a protocol using Microcon 30k centrifugal ultrafiltration units operated at 10,000 g.
In the ultrafiltration unit, 50 μg of total protein was mixed with 200 μL of 8 M urea in 0.1 M Tris/HCl, pH 8.5 (buffer 1), then centrifuged at 20 °C for 15 min.The eluates were discarded; 100 μL of buffer 1 was pipetted into the filtration unit, and the units were centrifuged again.
Flow-through from the collection tube was discarded, and a reduction buffer of 100mM TCEP in buffer 1 was added to achieve a final concentration of 10mM and incubated for 30 minutes at room temperature.Then, 50 μL of 0.05 M iodoacetamide in buffer 1 was added to the filters, and samples were incubated in darkness for 20 min.Filters were washed twice with 100 μL of buffer 1 followed by two washes with 100 μL of 25mM ammonium bicarbonate, pH 8.5 (digestion buffer-buffer 2).2.5ul of LysC (0.2 µg / µl) in 50 µL buffer 2 to filter and incubate for 4 h at 37ºC. 10 µL trypsin (0.1 µg / µl) in 300 µL buffer 2 was added to the filter and incubated overnight at 37ºC.Recovered peptides were filtered and collected in a separate tube.
Peptides were purified on C18 ZipTip columns.
2 ul of resulting tryptic peptides were analysed on a NanoAcquity-UPLC system (Waters) connected to a Q-Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer possessing an EASY-Spray nano-electrospray ion source (Thermo Fischer Scientific).The peptides were trapped on an in-house packed guard column (75 μm i.d.x 20 mm, Acclaim PepMap C18, 3μm, 100 Å) using solvent A (0.1% Formic Acid in water) at a pressure of 140 bar.The peptides were separated on an EASY-spray Acclaim PepMap® analytical column (75 μm i.d.× 50 mm, RSLC C18, 3 μm, 100 Å) using a linear gradient (length: 90 minutes, 3 % to 60 % solvent B (0.1% formic acid in acetonitrile), flow rate: 300 nL/min).The separated peptides were electrospray directly into the mass spectrometer, operating in a data-dependent mode using a CID-based method.Full scan MS spectra (scan range 350-2000 m/z, resolution 120000, AGC target 1e6, maximum injection time 250 ms) and subsequent HCD MS/MS spectra (AGC target 5e4, maximum injection time 100 ms) of 10 most intense peaks were acquired.HCD fragmentation was performed at 35 % of normalised collision energy, and the signal intensity threshold was kept at 500 counts.

Data Processing-Protein identification
Rationale -The protein analysis aimed to discover B. bacteriovorus proteins present at significant levels in the vesicle enriched preparations, which contained both predator and prey (E. coli or P. putida) materials, as they derived from invaded prey bdelloplasts.This proteomic approach was not an endpoint identification in itself.It was used to generate a list of potential candidate B. bacteriovorus proteins, which could then be tagged with mCherry and tested for cellular location microscopically to see if they did indeed reside in the vesicle in vivo during predation.
Data Processing -The analysis was performed with Peaks 8.5 software (Bioinformatics Solutions Inc).The raw MS file was first searched against the whole UniProt Database.
LysCTrypsin with a maximum of 3 missed cleavages and one unspecific end was selected as the protease.Carbamidomethylation (Cysteine) was set as a fixed modification, and Oxidation (Methionine) and Deamination (Asparagine, Glutamine) were set as variable modifications.
Precursor mass tolerance was set as 10 ppm.Fragment mass tolerances for HCD were set to 0.02 Da, respectively.All spectra were manually validated.All peptides present at -10lgP> 20, and spectra were manually checked, validated, or disqualified.PEAKS DB measures the quality of the Peptide-Spectrum Match (PSM) internally with a Linear Discriminative Function (LDF) score.To identify the protein, LDF considers the matching of fragment ions and spectrum peaks and the similarity between the de novo sequencing peptide and the universal database peptide, amongst other factors.The LDF score is converted to -10lgP to facilitate assessment.Peptides present at a p-value of 1% were selected for PSM validation with a Target Decoy PSM Validator node based on q-values at a 5% false discovery rate (FDR).Only validated peptides were used in protein database searches.A PEAKS PTM search was performed after a PEAKS DB search finished.PEAKS PTM analyses spectra with good de novo sequences that remain unidentified by PEAKS DB.The default setting for PEAKS PTM is to search with all the built-in modifications in the "Common" and "Uncommon" lists (more than 300 in-built modifications), which include all the natural modifications and mutations in the Unimod database.
To further investigate the protein associations, filtered data searches were conducted using Uniprot bacteria, the E. coli database and the Pseudomonas putida databases (considered prey "contaminant" proteins) and the B. bacteriovorus HD100 database for proteins from the predators.
The raw data are presented in Supplementary data.Supplementary Figure 9A shows the numbers of proteins identified as prey or Bdellovibrio in each prep.Supplementary Figure 9B shows the overlap in the datasets and identifies a core of 65 proteins in common between the two enrichment methods we took to be vesicle protein candidates for testing ( by mCherry tagging and microscopy).These data are presented in the "overlapping lists" tab in Supplementary data.The datasets had many contaminating prey proteins, as expected.Some differences between the Bdellovibrio protein candidates found in the datasets were likely due to the use of a modified protocol (as detailed in gradient vesicle enrichment section above) for vesicles from predation on E. coli data set 1 versus that used for the vesicle enrichment from predation on E. coli data set 2 & predation on P. putida dataset.
We didn't seek to resolve these differences, because we were using the process to identify putative Bdellovibrio protein candidates for validation via experimental testing, not as an experimental endpoint.We noted a strong enrichment (14 of the 65 proteins) of a group of proteins annotated as "cell wall anchor/YapH-like/phage-related tail fibre" proteins and concentrated attention on this group.
Other tested proteins with fluorescent tags discussed in this study permitted C-terminal fluorescent tags without mutation.
Fluorescence from S74 protein Bd3182-mCherry was not seen deposited in the predatory vesicle, but as a bright separate focus of Bd3182-mCherry fluorescence deposited elsewhere distally from CpoB in the prey bdelloplast, and only clearly seen at a later stage, approximately 120-180 minutes into predation (red focus,Fig. 2A Supplementary Data Figs. 11 and 15).In a Bd3182(S782A)-mCherry strain (Supplementary Data Fig 15), engineered to substitute the active site serine residue (proteolysis activity via Ser782 can be confidently predicted from UniProt annotation, and is confirmed in our structural studies herein), in-vivo fluorescent mCherry foci were scarcely detectible.These data suggest that S74-mediated cleavage of the chaperone domain happens around 120-180 minutes into predation to allow access to the bdelloplast binding site of Bd3182 only at this point and serendipitously allows unrestricted fluorescence of the chaperone-bound mCherry.Linkage of the mCherry tag at the C-terminal end of the chaperone infers that we follow the released chaperone domain in the wildtype S74 MAT proteins.This potential complication is not an issue for the NS74 MAT members as they do not have the protease site to allow auto cleavage.
We expanded our fluorescent tagging and microscopic experimentation into other protein family members that had not come up in the proteome of our initial vesicle preparation, mindful that the relative level of expression of "cell wall anchor/YapH-like/phage-related tail fibre" proteins was only being tested proteomically at the end of predation (300 minutes and later into predation) by our sampling of vesicles in the lysed prey debris.Base strain and plasmid details are shown in Supplementary Table 7.
NS74 proteins Bd1334, Bd2734 and Bd2740 were chosen as they represented different protein family subgroupings, with potentially different functionalities (as identifiable from differing C-terminal folds at the end of the fibre, details of these are provided in the main text).We found that Bd2740mCherry did locate to the CpoBBd0635-containing vesicle, deposited during predation, while Bd1334mCherry and Bd2734mCherry fluorescence was diffuse throughout the B. bacteriovorus cell (Fig. 2A and Supplementary Data Fig. 11).Frequencies of colocalisation of Bd2133, Bd2439 and Bd2740 with CpoB throughout the timecourse of predation are presented in Supplementary Data Fig. 12.      Homologous genes to all adhesins found in strain HD100 are shown for strain 109J.All HD100 genes were blasted against the 109J genome using tblastn.Alignments were made using Clustal Omega, with the sequence identity for each shown.Interestingly, although most adhesins have homologues with high sequence identity, bd2439 is lost in 109J (highlighted red).

Supplementary Table S5-Primers and Plasmids used in this study
CpoB Phyre2 output shows that the predicted structure of CpoB from B. bacteriovorus HD100 (query sequence) is highly conserved and maps to the E. coli K12 CpoB structure (template sequence) with a high confidence score (99.8).Alpha helices are in green, T is a hydrogen-bonded turn, and S is a bend.Supplementary Figure 2. Distribution of CpoB-mCherry patterns of B. bacteriovorus HD100 attached to prey cells compared to those entering prey cells.Four independent data points were obtained by using 15 min and 20 min timepoint data from two independent experiments.Means and independent data points are presented.Error bars are SEM.* denotes significance p<0.05 by the two-tailed Mann-Whitney test Exact P = 0.286 for all three.Supplementary Figure 3. CpoB-mCherry Distributions.Distribution of fluorescent CpoB-mCherry patterns in wild type B. bacteriovorus HD100 attached to (15-20 min), entering (20-25 min), or fully entered (25-30 min) into prey cells.Means and data points of two independent experiments are presented.Error bars are SEM.Values of n (Bdellovibrio cells analysed) are shown for each experiment and time point.Supplementary Figure 4. Distribution of fluorescent CpoB-mCherry patterns in mutant B. bacteriovorus Δbd0886Δbd1176 (double LD-transpeptidase deletion mutant background).Mutant strain attached to (15-20 min), entering (20-25 min), or fully entered and establishing within the bdelloplast (25-45 min) prey cells.These data are to validate the use of this strain versus data in Supplementary Figure 3 for wild-type B. bacteriovorus (in Supplementary Figure 5) to show up the HADA-labelled porthole in a bdelloplast background with less additional lateral incorporation of other D-amino acids by LD-transpeptidation). Means and data points of two independent experiments are presented.Bdelloplasts were scored for when a focus of CpoB-mCherry spot appeared to be co-localised with a HADA spot (HADA coincidence).Error bars are SEM.Values of n (Bdellovibrio cells analysed) are shown for each experiment and time point.Supplementary Figure 5. CpoB: HADA Co-localization.A Phase and epifluorescence microscope images demonstrating a fluorescent CpoB-mCherry focus (red) co-locates in the same place as the HADA "porthole" focus (blue) in the wall of a bdelloplast formed by predation by B. bacteriovorus Δbd0886Δbd1176 (CpoB-mCherry) and pulse-labelled with HADA.Individual phase and fluorescence channels and merges are presented.Images are representative of two independent experiments.Scale bars are 2 µm.B Plot of the number of bdelloplasts analysed in two independent experiments that displayed a focus of CpoB-mCherry in the same position as a focus of HADA.Means and independent data points are presented.Supplementary Figure 6.Bdelloplast CpoB-mCherry: HADA Relationship.A Composite phase and epifluorescent microscope image showing a bdelloplast with co-incident CpoB-mCherry and HADA foci.The scale bar is 1 µm.B Line drawn through the bdelloplast traversing the co-incident foci.C Plot of fluorescence values along the line drawn in B showing co-incident peaks (arrow) of CpoB-mCherry (green) and HADA (blue).D Plots, as in C, scaled individually.An example typical of 40 analysed bdelloplasts is shown from two independent experiments.Supplementary Figure 7. Co-localisation Analysis for HADA and CpoB mCherry protein fluorescence.Analysis was carried out using the coloc2 plugin for ImageJ by either choosing an ROI around the whole bdelloplast (1) or an ROI around the CpoB-mCherry focus (2).A control ROI was made by moving the bdelloplast ROI to a nearby background region (3).Coloc2 was run with Manders' correlation algorithm and Costes' significance test with 1000 randomisations.An example typical of 40 analysed bdelloplasts is shown.The table shows the correlation R and significance P for these different analyses.Both methods at all timepoints demonstrated significant colocalisation (P>0.95), but never for the control.
image correlation coefficient R and averaged Costes P calculated by 1000 rounds of Costes randomisation: Supplementary Figure 14.Positions of extracellular immunofluorescence detected with FITC anti-mCherry on B. bacteriovorus cells which are invading E. coli prey.Cells attached to prey at 20 minutes post-mixing of predator and prey were manually scored for immunofluorescence spot position.Representative merges of epifluorescence and phase are shown.Scale bars are 2µm.
single-crossover, full length Bd0635-mCherry fusion and a double-crossover, full length Bd2734-mTeal fusion This study B. bacteriovorus HD100 Bd1334mCh_SXO plus Bd0635mT_DXO HD100 containing a single-crossover, full length Bd1334-mCherry fusion and a double-crossover, full length Bd0635-mTeal fusion This study containing an in-frame unmarked deletions of both bd0886 and bd1176 Kuru et al., 2017 B. bacteriovorus HD100 bd1334 B. bacteriovorus containing an in-frame unmarked deletion of bd1334 This study B. bacteriovorus HD100 bd2133 B. bacteriovorus containing an in-frame unmarked deletion of bd2133 single-crossover, full length Bd0064-mCherry fusion and a double-crossover, full length Bd2734-mTeal fusion This study